Toxoplasma gondii is an important opportunistic pathogen, affecting up to one&#8208;third of untreated AIDS patients worldwide. The parasite possesses a unique and essential organelle called the apicoplast, which is genetically and functionally related to chloroplasts. Because the human host lacks a similar organelle, the apicoplast is an important potential target for development of anti&#8208;parasitic agents. Little is known about the biogenesis of this organelle, which is bounded by four membranes. Until now, only targeting to the apicoplast lumen has been explored. Such proteins are targeted courtesy of a signal sequence that allows entry into the secretory system and a transit peptide that routes them from the ER to the plastid. We have presented provocative evidence that the latter step occurs in a Golgiindependent manner. In the past grant period we identified two apicoplast membrane proteins (a transporter and a protease), both of which lack canonical targeting sequences. In contrast to luminal proteins, these proteins show cell cycle dependent localization to the apicoplast. Because of these differences as well as the distinct ultimate destinations, the routes and sequences that mediate targeting of non&#8208;luminal proteins likely involve novel elements. This proposal outlines experiments aimed at characterizing a set of proteins localized to various apicoplast compartments, and will dissect the mechanisms and sequences important for the proper localization of these proteins. We will also determine whether targeting of apicoplast membrane proteins is assisted by molecules known to be involved in ER exit processes or ER to Golgi trafficking. These studies will expand our understanding of the biogenesis of this unique organelle and provide additional information concerning its function.